TRANSFORMATION OF NIH3T3 FIBROBLASTS BY THE C-KIT RECEPTOR TYROSINE KINASE - EFFECT OF RECEPTOR DENSITY AND LIGAND-REQUIREMENT

Ectopic expression of the normal murine receptor tyrosine kinase, c-Ki t, in NIH3T3 cells induced many phenotypic changes characteristic of t ransformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice, Although transformation was largely d ependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in the absence of added factor, and this could not be inhibited by neutralising antibodies or by SLF anti-sense mRNA, Clo nes from factor-independent colonies in semi-solid agar displayed a na rrow range of c-Kit surface protein levels (4.3-6.4 x 10(4) receptors/ cell) which was relatively high compared with the pool from which they were derived, Analysis of a larger series of random clones derived fr om adherent cultures expressing different levels of c-Kit demonstrated a positive correlation between SLF-dependent, anchorage-independent g rowth and c-Kit protein and mRNA expression levels (respectively, R-S = 0.58, P < 0.01; and R-S = 0.53, P < 0.01) with consistent colony for mation observed with clones having > 2.5 x 10(4) receptors/cell, Inter estingly, two of the three clones expressing the highest levels of c-K it protein and mRNA produced few or no colonies in the presence or abs ence of SLF, Sequential overexpression of human c-KIT in NIH3T3 cells using a dihydrofolate reductase (DHFR)-encoding vector and gene co-amp lification through methotrexate selection, which resulted in pools exp ressing up to 1.5 x 10(5) receptors/cell, confirmed that high receptor densities resulted in a decrease in colony numbers, Thus, analysis of clonal and selected populations has indicated that an optimal level o f c-Kit is required for transformation of NIH3T3 cells in the presence of SLF, and that some ligand-independent transformation occurs.